Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins |
| |
Authors: | Izabela Sokolowska Armand G Ngounou WetieUrmi Roy Alisa G WoodsCostel C Darie |
| |
Institution: | Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY 13699-5810, USA |
| |
Abstract: | We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC–MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed. |
| |
Keywords: | ZP zona pellucida SP signal peptide FLCS furin-like cleavage site TM transmembrane IgG-HC protein immunoglobulin heavy chain protein IgG-Fc-ZP3E7 protein the protein product of the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 ZP3E7 polypeptide that corresponds to exon 7 from ZP3 SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis WB Western blotting ECL enhanced chemiluminescence MS mass spectrometry LC&ndash MS/MS liquid chromatography tandem mass spectrometry m/z mass/charge CID collision-induced dissociation DDA data dependent analysis IDA information dependent analysis (DDA with inclusion list) TIC total ion chromatogram XIC extracted ion chromatogram |
本文献已被 ScienceDirect 等数据库收录! |
|